In the current state of the art, the measurement of concentrations of various ions of a medium is performed in highly varied ways. One of the most widely used techniques is the use of test strips. These test strips are strips of paper with different areas which become coloured in contact with aqueous solutions, taking on different colours depending on the concentration of specific ions of the measurement solution. To identify the concentration of ions of the solution, after wetting the strip therewith, the user must compare the colours obtained with those of a table provided by the manufacturer. The result of this measurement technique greatly depends on the correct manipulation by the user and factors such as: the presence of proteins in the samples, the reaction time of the strip with the samples, or the homogeneity of the samples. An incorrect manipulation generates many false results (positive and negative). Furthermore, it is generally considered that the resolution of this technique is of 0.5 units, for the specific case of pH measurement, which lacks sufficient diagnostic value to take clinical decisions in some biomedical applications such as in Urolithiasis (Kwong T. et al. “Accuracy of urine pH testing in a regional metabolic renal clinic: is the dipstick enough?. Urolithiasis 2013).
The standard measurement technique of ion concentration is atomic absorption. However, this technique requires a complex installation and its miniaturization is not feasible.
Ion Selective Electrodes (ISEs) are used for simpler measurements in terms of equipment and are less expensive. These electrodes have a selective membrane so that, by the exchange or interaction of the solution ions with the membrane, the ion activity becomes an electric potential. The selective membrane may be of several types, of glass, crystalline or based on ion-exchange compounds. The latter have a polymer (e.g. polyvinyl chloride, PVC) which immobilizes the ion selective compound. The measurement of the electric potential of the ISEs requires the use of a reference electrode, which is frequently integrated in the very body of the ISE (combined electrodes). The reference electrode is generally a metal electrode immersed in a reference solution which is in turn connected to the solution to be measured through a liquid bond. The main characteristic of the reference electrode is that its potential, i.e. the potential between the interior of the metal and the inside of the solution wherein it is immersed, does not depend on the composition of said solution. The reference electrodes usually have losses of reference solutions through the liquid bond, so that a periodic refilling thereof is required.
To obtain precise measurements, these electrodes require a prior calibration which consists of the measurement of the potential generated when the electrode is immersed in a known ion concentration solution. These electrodes form part of an instrument, which in the case of the pH is known as pHmetre, which is not cheap to manufacture, nor is it portable or autonomous and it requires specific maintenance and cleaning conditions for its correct conservation. The result of this measurement technique also depends on the correct manipulation by the user (who must be suitably trained for said purpose). An incorrect manipulation or conservation of the electrodes may give rise to false results.
Another type of sensors used for this class of measurements are the ISFET (ion-selective field effect transistor)-type sensors. These are devices manufactured using microelectronic technology. The potential of the solution (which is the transistor gate potential) is controlled by a reference electrode such as those used for the measurement with ISE-type electrodes. The ISFET is a field effect transistor whose threshold voltage varies with the ion concentration of the solution in contact with its gate dielectric. For many types of dielectrics (SiO2, Si3N4, Al2O3, Ta2O5, ZrO2), the variation of threshold voltage of the ISFET mainly depends on the H+ ion and, therefore, it is used as pH sensor. To make sensors of other ions based on the ISFET, an additional layer called selective membrane as disclosed in U.S. Pat. No. 5,250,168 is deposited on the gate dielectric layer. Depending on the membrane deposited, the ISFET would function as a sensor for specific ions or others. The measurement with these sensors consists of recording the changes in threshold voltage of the field effect transistor, which are proportional to the changes in ion concentration that one wants to measure. A way of measuring the changes in threshold voltage of the ISFET is using a circuit which polarizes the device with a constant drain current and a constant source drain voltage. In this way, the changes in gate voltage the circuit applies to maintain said polarization are equal to the threshold voltage changes suffered by the ISFET. Therefore, the gate voltage applied by the circuit is taken as output signal.
Both the measurement systems based on ISE electrodes and those based on ISFET require a reference electrode to be able to measure the ions. This makes them expensive and requires periodical maintenance. In 1978, a solution was disclosed for pH measurement with ISFET-type devices without reference electrode (P. A. Comte and J. Janata, “A field effect transistor as a solid-state reference electrode”, Analytica Chimica Acta) which consisted of the differential measurement of an ISFET and a REFET. In this case the REFET is formed by an ISFET the gate whereof is kept exposed to a constant pH. The differential measurement consists of measuring the threshold voltage of both devices using a single electrode immersed in the solution as terminal gate and obtaining the answer as subtraction of the two values obtained. The REFET gate is maintained exposed to a constant pH by the incorporation of a microreservoir filled with reference solution (internal solution). Said microreservoir is connected to the exterior via a microchannel which acts as liquid bond, so that the difference in potential between the external solution and the reference solution is small and is not greatly influence by the pH or the concentration of other ions in the external solution. In this way, the changes in potential which occur between the electrode and the solution are transferred to both threshold voltage values, and therefore have no impact on the differential value (they are cancelled in the subtraction operation). For this reason, the different measurement system can be implemented with any conductor electrode, without the need for it to be reference. Given that the REFET is exposed to a constant pH solution, the variation in the differential value shall be equivalent to the ISFET's response to the change in pH. However, the way of manufacturing the REFET described by Compte and Janata is difficult to automate and would therefore not make it possible to manufacture the sensors at a cost greatly less than those of the ISFETs with reference electrode, which would not allow its price to be accessible to the general public. Furthermore, in the design of the ISFET-REFET sensor described by Compte and Janata, the REFET's microreservoir is constructed with an epoxy resin. This microreservoir, once the resin has been cured, is filled with an agarose gel prepared in a buffer solution. Subsequently, a glass capillary is introduced, which acts as a microchannel, in the agarose gel and the microreservoir is sealed with a layer of epoxy resin. Thus, the sensor is stored dry; the buffered solution of the microreservoir is slowly evaporated through the microchannel, being replaced by air. The presence of air interior the microreservoir prevents it functioning correctly when it is used after an extended time has passed of immersion in aqueous solution. This is due to the fact that the filling with water, as well as the necessary diffusion of the trapped air to the exterior, is solely performed through the microchannel, which is not filled with hydrogel. Furthermore, the lifetime of this type of sensor depends on the volume of the microreservoir and on the dimensions of the microchannel which connects it with the exterior, since the reference solution in the microreservoir shall be diluted and contaminated through the microchannel, so that the error in the measurement may progressively increase a measurement that the pH of said solution varies with respect to its original value. For this reason, it is considered a sensor with a short lifetime.
Document EP 85200263 discloses a sensor wherein use is made of two ISFET sensors, one of which is found interior a conduit wherethrough the reference solution flows. In this way, said ISFET is always in contact with an uncontaminated solution. However, for this it is necessary to incorporate in the sensor a reference solution injection system as well as the means of supply of the injection system which make the solution described more complex and expensive.
Thus the state of the art has the following associated problems: the test strips are imprecise; the glass electrodes are expensive, fragile, require maintenance and cannot easily be miniaturized; the current ISFET and ISE-type sensors are miniaturizable but are expensive and require maintenance as they must be used with a reference electrode; the ISFET-REFET sensor proposed by Compte and Janata is expensive to manufacture and has a short lifetime; and the sensor disclosed in EP 85200263, in addition to concerning two ISFET transistors, has a greater cost and complexity due to the need to have a reference solution injection system.